CN109845440B - Method for promoting soil nitrate reduction by using iron film at rice root - Google Patents

Abstract

The invention discloses a method for promoting soil nitrate reduction by a rice root iron film, wherein the rice iron film can enhance the reduction of nitrate, and is coupled with the iron oxidation reduction process, so that N is added₂Discharging of (3); the invention can be rich inThe method integrates denitrifying microorganisms and iron redox microorganisms of soil, can enhance the nitrate reduction of crop microorganisms, and couples the redox of iron, thereby reducing the risk of nitrate leaching loss, and is an environment-friendly method under in-situ conditions, and has strong practical operability.

Description

Method for promoting soil nitrate reduction by using iron film at rice root

Technical Field

The invention belongs to the technical field of soil improvement, and particularly relates to a method for promoting soil nitrate reduction by using an iron film at the root of rice.

Background

Nitrogen is one of the most important elements lost by leaching in farmland. The nitrogen in the soil exists in an organic state and an inorganic state, wherein the organic nitrogen comprises two forms which are easy to hydrolyze and difficult to hydrolyze, and the inorganic nitrogen comprises free nitrogen, ammonium nitrogen, nitrate nitrogen and nitrite nitrogen. Most of the soil is colloid with negative charges, so that a large amount of ammonium nitrogen is easy to be adsorbed, and nitrate nitrogen is difficult to be adsorbed. Therefore, under the action of rainfall and irrigation, most of nitrogen in the soil is leached out by soluble nitrate nitrogen. Therefore, under the condition of sufficient water management, the control of the content of nitrate nitrogen in soil becomes a main factor for controlling the leaching loss of nitrogen. At present, the mode of avoiding the loss of soil nitrogen is mainly to add a nitrification inhibitor into soil to inhibit the nitrification in the soil, thereby reducing the content of nitrate nitrogen. However, the nitrification inhibitors are chemical substances, and are easy to cause secondary pollution. The technology for influencing the reduction of the nitrate in the soil by utilizing the iron membrane of the plant root is not reported, and the method for enhancing the reduction of the nitrate by developing the technology which is environment-friendly and strong in operability is of great significance to the technology for removing the nitrate in the soil under the in-situ condition.

Disclosure of Invention

One of the objects of the present invention is to provide a method for promoting the reduction of nitrate in soil.

It is another object of the present invention to provide a method for enriching nitrate-reducing microorganisms in soil.

It is yet another object of the present invention to provide a method for enriching iron redox microorganisms in soil.

The technical scheme adopted by the invention is as follows:

a method for promoting reduction of nitrate in soil is characterized in that after an iron film at the root of rice is induced to form, the rice is transplanted into the soil for culture, and the soil is flooded for 5-9 mm, wherein the flooding time is 9-10 days.

Further, the rice seeds were treated with 10% H₂O₂And (3) soaking the seeds in the solution for 10-15 min for disinfection, thoroughly cleaning the seeds with water, soaking the seeds in the water, and accelerating germination in an incubator.

Further, transplanting the two-leaf-period seedlings with the accelerated germination into a rice full nutrient solution for culturing for 18-24d, wherein the nutrient solution is changed once every 3d, and the pH value is 5.5-5.8.

Further, the method for forming the iron film on the rice root comprises the following steps: putting the rice seedlings in 70-80 mg L^-1Performing induction culture in a ferrous sulfate solution for 40-48 h.

Further, the rice is a seedling in a four-leaf stage.

Further, the soil contains denitrifying microorganisms and/or iron redox microorganisms coupled to denitrification.

A method for enriching microorganisms for reducing nitrate in soil comprises the steps of inducing the formation of an iron film at the root of rice, planting the rice in the soil, and flooding the soil by 5-9 mm; the flooding time is 9-10 days.

Further, after the rice is transplanted into the soil, the soil is flooded after being cultured for 5 days.

Further, the method for treating the flooding comprises the following steps: and (3) adding water into each pot with the soil dry weight of 1.5kg and keeping the water flooding layer to be 5-9 mm.

Further, the nitrate-reducing microorganisms include denitrifying microorganisms and iron redox-related microorganisms.

Further, the denitrifying microorganism includes Pseudomonas, Thoibacillus, Thermomonas, Bacillus, Candidatus Nitrososphaera.

Further, the iron redox-related microorganism includes Thermomonas, Rhodobacter, Pseudomonas, Desloromonas, Thiobacillus, Clostridium, Desulfovibrio, Alkaliphilus, Anaeromyxobacterium, Desulfotomacuum, Alicyclobacillus, Bacillus.

A method for promoting denitrification in soil to couple iron oxidation reduction comprises the steps of inducing the formation of an iron film at the root of rice, and then planting the rice in the soil, wherein the soil contains iron oxidation reduction microorganisms capable of being coupled with nitrate reduction.

A method for enriching soil capable of coupling nitrate reduction-related iron-oxidizing reducing microorganisms including Thermomonas, Rhodobacter, Pseudomonas, Deschloromonas, Thiobacillus, Clostridium, Desulfovibrio, Alkaliphilus, Anaeromyxobacter, Desutomaculum, Alicyclobacillus, Bacillus, and transplanting rice into soil after inducing iron film formation in rice roots.

The invention has the beneficial effects that:

(1) the rice iron film can enhance the reduction of nitrate, N₂O and N₂And couples the process of iron oxidation reduction. Water-soluble N in soil transplanted with rice with iron film (IP)₂O and reactor headspace N₂The concentration of O is obviously higher than that of the soil of a control group (CK), and simultaneously, the content of nitrate nitrogen in the soil is reduced, so that the nitrate in the soil can be used for strengthening the nitrate reduction process through denitrification;

(2) the method is an environment-friendly technology under in-situ conditions, has strong practical operability, can enhance the microbial nitrate reduction of crop roots, and couples iron oxidation reduction, thereby reducing the risk of nitrate leaching loss;

(3) the invention can enrich and reduce the microorganism of nitrate;

(4) the invention is suitable for the soil with low content of iron oxide and excessive nitrate nitrogen fertilization.

The method is a mode for effectively reducing the nitrate pollution of underground water by enhancing the nitrate reduction of the soil at the roots of plants, and the iron film at the roots of rice is an iron nutrition bank and mainly composed of amorphous iron oxide. To date, there is still a lack of information on how the iron film of plant roots affects the reduction of nitrate in soil. Such information is essential for the technology of soil nitrate removal under in situ conditions.

Drawings

FIG. 1 is a photograph of a rice blank (CK) and a rice plant in which iron film (IP) formation has been induced;

FIG. 2 shows the iron membrane content changes (a), N, after the rice plants of the IP group and CK group were transplanted into the soil and flooded with water₂O discharge rate (b) and soluble N₂O concentration (c) (n ═ 3);

FIG. 3 is a graph showing the relative abundance (n-3) of non-metric multidimensional scale (NMDS) analysis of soil and root microflora (a), non-metric multidimensional scale (NMDS) analysis of soil and rhizosphere soil microflora (b) and genera (c) after the rice plants of IP group and CK group are transplanted into soil and flooded with water; in the graph (c), red represents iron-reducing bacteria, and black represents iron-oxidizing bacteria;

FIG. 4 is a plot of soil and rhizosphere soil microbial communities;

FIG. 5 shows the headspace N of rice plants with IP and CK groups placed in a hydroponic liquid system₂O concentration (a), headspace N₂Concentration (b), head space N treated by CK after ferrous ion is added into solution₂O concentration (c), headspace N of IP treatment after EDTA addition in solution₂O concentration (d), ferrous ion concentration (e) in the IP treatment in different solutions and nitrate nitrogen concentration (f) in the solution (n-3).

Detailed Description

A method for promoting nitrate reduction in soil comprises the steps of transplanting rice into soil for culture after inducing iron films at rice roots to form, and flooding the soil for 5-9 mm, wherein the flooding time is 9-10 days.

Preferably, the rice seeds are treated with 10% H₂O₂And (3) soaking the seeds in the solution for 10-15 min for disinfection, thoroughly cleaning the seeds with water, soaking the seeds in the water, and accelerating germination in an incubator.

Preferably, the seedlings in two leaf periods of pregermination are transplanted into the rice total nutrient solution for culturing for 18-24d, the nutrient solution is changed once every 3d, and the pH value is 5.5-5.8.

Preferably, the method for forming iron film on rice rootComprises the following steps: putting the rice seedlings in 70-80 mg L^-1Performing induction culture in a ferrous sulfate solution for 40-48 h.

Preferably, the rice is young seedlings in the four-leaf stage.

Preferably, the soil contains denitrifying microorganisms and/or iron redox microorganisms coupled to denitrification.

A method for enriching microorganisms for reducing nitrate in soil comprises the steps of inducing the formation of an iron film at the root of rice, planting the rice in the soil, and flooding the soil by 5-9 mm; the flooding time is 9-10 days.

Preferably, after the rice is transplanted into the soil, the soil is flooded after 5 days of culture.

Preferably, the method for treating flooding comprises the following steps: and (3) adding water into each pot with the soil dry weight of 1.5kg and keeping the water flooding layer to be 5-9 mm.

Preferably, the nitrate-reducing microorganisms include denitrifying microorganisms and iron redox-related microorganisms.

Preferably, the denitrifying microorganisms include Pseudomonas, Thoibacillus, Thermomonas, Bacillus, Candidatus Nitrososphaera.

Preferably, the iron redox-related microorganism includes Thermomonas, Rhodobacter, Pseudomonas, Desloromonas, Thiobacillus, Clostridium, Desulfovibrio, Alkaliphilus, Anaeromyxobacterium, Desulfotomacuum, Alicyclobacillus, Bacillus.

A method for promoting denitrification coupling iron oxidation reduction in soil is characterized in that after an iron film at the root of rice is induced to form, the rice is planted in the soil, and the soil contains microorganisms capable of reducing nitrate coupling iron oxidation reduction.

A method for enriching soil capable of coupling nitrate reduction-related iron oxide reduction microorganisms including Thermomonas, Rhodobacter, Pseudomonas, Deschloromonas, Thiobacillus, Clostridium, Desulfovibrio, Alkaliphilus, Anaeromyxobacter, Desutomaculum, Alicyclobacillus, Bacillus, and transplanting rice into soil after inducing iron film formation in rice roots.

The present invention will be further described with reference to the following examples.

Example 1 method for regulating and controlling nitrate in soil by using iron film

In the study, soil is collected in an area where nitrogen fertilizer is applied for a long time at test station of goldenrain city, academy of sciences, China (37.90 degrees north latitude, 114.67 degrees east longitude, elevation 50 m). Surface soil was collected at 5 different points in the test area. The soil was first air dried and sieved using a sieve with a 2mm aperture. The soil has a clay content of 7.3%, a particle content of 79.8% and a sand content of 12.3%. The organic matter content of the soil is 1.9g kg^-1pH 7.1, nitrate NO₃N31.3 mg kg^-1And a soluble organic carbon concentration of 4.1mg kg^-1. In the experiment, the nitrate nitrogen concentration of the soil reaches 43-47 mg kg due to the use of the fertilizer^-1. FIG. 1 is a graph of rice blank Control (CK) and rice in which iron membrane formation (IP) has been induced.

(1) The rice seeds of the variety named Nipponbare are treated with 10% H₂O₂Soaking in the solution for 10min for sterilization, thoroughly cleaning with sterile water, soaking rice seeds in sterile water, and performing germination acceleration in an incubator at 30 deg.C for 48 h. After the seeds germinate, transplanting the seedlings in the two-leaf period into the rice full nutrient solution for culturing for 21 days, wherein the nutrient solution is changed every 3 days, and the pH value is 5.5-5.8. The culture conditions of the artificial climate box are as follows: the time and temperature of the day are 14h and 28 ℃; the time and temperature of the night was 10h, 25 ℃, and the same culture conditions were used for subsequent experiments.

(2) After the rice seedlings grow for 4 weeks, selecting four-leaf stage rice seedlings with consistent growth vigor, dividing the four-leaf stage rice seedlings into two groups, transferring the two groups to water for culturing for 24 hours, wherein the seedlings of the control group are continuously cultured in the water for 48 hours (CK), and the seedlings of the experimental group are transferred to a medium containing 80mgl^-1Is cultured in ferrous sulfate solution for 48h (IP). Transplanting the rice of CK and IP group into cylindrical pot (phi 22 × 35cm) (24 pots are prepared for CK and IP treatment, 6 rice seedlings are loaded in each pot), inserting into soil to fix roots in the soil, and performing soil flooding treatment after adapting to 5d, wherein the height of water covered on each pot is 5mm, and the flooding treatment is performedThe time period was 10 d.

(3) Sealing the pot with rubber stopper, screw and pot cover, and measuring N by gas chromatography₂An O discharge rate; opening the can lid, and measuring the soluble N with microelectrode₂The concentration of O. Then, for NH in the soil₄ ⁺、NO₃ ^-、NO₂ ^-And analyzing the DOC content; finally, the distribution of microbial community structures in soil, rhizosphere soil and plant roots is determined.

(4) To determine the major pathway of nitrate decomposition, rice seedlings from CK and IP groups were placed in anaerobic flasks and NH was added separately₄ ⁺And NO₃ ^-As a nitrogen source of the culture solution, the anaerobic flask was alternately evacuated five times (0.1kPa) to replace the volume with He/O₂Mixed gas (79/21%, 101.3kPa) process. All the anaerobic bottles are placed at 28 ℃ in the daytime and 25 ℃ in the evening, the bottles are alternately treated in light and dark for 240h, the time points of 0, 6, 11, 24, 30, 48, 72, 96, 103, 120, 144, 192h and 240h of treatment are selected, and 3 anaerobic bottles are randomly selected in the IP group and the CK treatment group respectively. Sampling the reactor headspace with an automatic sampling analysis system to determine N₂O and N₂And (4) concentration.

(5) To determine the role of Fe (II) in the major nitrate decomposition pathway, rice roots of CK and IP groups were placed in anaerobic flasks and NO alone was added₃ ^-EDTA and Fe (II) as nitrogen sources of the culture solution were added to the respective treatments, and the anaerobic flask was alternately evacuated five times (0.1kPa) and replaced with high-purity helium (99.9999%, 120kPa) (the content of oxygen in the soil below the root was extremely low, almost under anaerobic conditions, and in order to simulate this, helium gas which is an inert gas was used to replace the oxygen-containing air in the flask). The headspace air was adjusted to 101.3kPa after the last fill with helium (the pressure was adjusted to keep the pressure in the reactor consistent with the air pressure). All the anaerobic bottles are placed at 28 ℃ in the daytime and 25 ℃ in the evening, the bottles are alternately treated and observed for 168 hours in light and dark, the time points of 0, 5, 7, 12, 24, 30, 36, 48, 72, 120 hours and 168 hours of treatment are selected, and 3 anaerobic bottles are randomly selected in the IP group and the CK treatment group respectively. By self-energizingThe dynamic sampling analysis system samples the headspace of the reactor to determine N₂The O concentration.

The measuring method of the corresponding index comprises the following steps:

1. NH in soil₄ ⁺,NO₃ ^-And NO₂ ^-Method for measuring content and DOC (soil soluble organic carbon)

N in headspace gas₂After the O concentration is determined, leaching inorganic nitrogen in the soil by using 1M KCl, and performing indophenol blue colorimetry, dual-wavelength spectrophotometry and nitrite spectrophotometry on NH in the soil₄ ⁺、NO₃ ^-And NO₂ ^-Carrying out measurement; and (3) determining the DOC content in the soil by utilizing a total organic carbon analyzer TOC instrument.

2. Determination of iron film content, microbial community structure and soluble N in rice roots₂The concentration of O varies from the plant rhizosphere to the concentration of the soil in the horizontal direction.

In the process of rice culture, a abandon sampling method is adopted, and the content of the iron membrane at the root of rice is measured by a DCB method (a sodium citrate-sodium bicarbonate-sodium dithionite method).

After 240h of flooding treatment, N is used₂Solubility N of O microelectrode to soil in horizontal direction from rhizosphere soil to soil body soil₂The spatial distribution of the O concentration was determined. The microsensors were inserted into the soil to a depth of 3.5cm and the data was recorded on a computer by sensor tracking software. For solubility N₂After the O concentration was measured, the soil was divided into two parts (rhizosphere soil and soil body soil). Then, the soil attached to the roots of the plants was washed by an ultrasonic method, and DNA of the roots of the plants and the soil attached to the roots of the plants were extracted, and the microbial community structure was measured and analyzed.

Total DNA of microorganisms in soil is extracted by Fast DNA SPIN Kit of MP Bio company, and then sequencing detection is carried out. The quality and quantity of the extracted DNA was checked by NanoDrop. The extracted DNA was analyzed for microbial community structure by 16S rRNA gene sequencing.

For microbial community structure analysis, the gene of interest was amplified using 515f/907r primers, the primer sequences were as follows:

515f:5'-GTGCCAGCMGCCGCGGTAA-3'(SEQ ID NO：1)；

907r:5'-CCGTCAATTCCTTTRAGTTT-3(SEQ ID NO：2)。

measurement results of respective indices:

NO in soil after transplanting CK group and IP group rice₃ ^-、NO₂ ^-、NH₄ ⁺Discharge N₂O and soluble N₂Change in O content

IP group treatment significantly increased soluble N in soil compared to CK group₂Concentration of O and N₂The emission rate of O (see FIG. 2), but the IP group reduced the nitrate concentration (see Table 1), and the results preliminarily confirmed that the iron film can significantly enhance NO in soil₃ ^-And (4) reduction process. The iron content of each gram of dry heavy root in the experiment is the iron film content, the iron oxide of the plant is consumed by the outside along with the growth of the plant root, and the like, and the iron film content of the root treated by the IP group in the experiment is reduced from 5.74 to 2.88mg g^-1The dry weight of the root is ten times (0.28-0.76 mg g) of the iron film content of the CK group treatment^-1Root dry weight), indicating that iron films are a major factor affecting nitrate reduction.

TABLE 1 NO in pots before and at 140h of planting₃ ^-、NO₂ ^-、NH₄ ⁺Change of DOC average content (n ═ 3)

Note: the different letters (a, b) indicate that there is a significant difference between the control and experimental groups (0.01< P < 0.05).

Secondly, after the CK group and IP group paddy rice are transplanted into the soil, the relative abundance of denitrifying microorganisms and iron oxidation reduction microorganisms of the soil is changed

Compared with CK treatment, IP treatment significantly changed the typical bacterial growth in the iron redox cycle in rice rhizosphere soil and rootsThe structure of the object community increases the relative abundance; through non-metric multidimensional scale (NMDS) analysis, it was found that there was a significant difference in rhizosphere soil and root microbial communities between the IP group and the CK group (see (a) (b) of fig. 3), some bacteria were also involved in soil denitrification, such as Pseudomonas, Thiobacillus, Thermomonas, Bacillus, Candidatus nitrosolpora (see fig. 4); participating in iron redox, such as Thermomonas, Rhodobacter, Pseudomonas, Dechloromonas, Thiobacillus, Clostridium, Desulfovibrio, Alkaliphilus, Anaeromyxobacter, Desulfomammium, Alicyclobacillus, Bacillus (see FIG. 3 (c)). These results indicate nitrate reduction and N after IP treatment₂The increase in O emissions may be related to the iron redox cycle of the microorganisms.

Thirdly, after the CK group and the IP group of rice are placed in the hydroponic liquid, N₂O、N₂Fe (II) and NO₃ ^-Change in concentration of

To determine the main pathway of nitrate decomposition and the role of fe (ii) in the main pathway of nitrate decomposition, experiments with hydroponics were performed. The results show that treatment of the IP group is significantly increased with NO₃ ^-N as the sole nitrogen source₂O-emission with simultaneous increase of N₂Emission of (2), reduction of NO₃ ^-In concentration of NH₄ ⁺This difference was not evident in the solutions (see fig. 5a, 5b and 5 f). The results prove that nitrate reduction is mainly performed by a denitrification way, and further prove that the iron film can obviously enhance NO₃ ^-And (4) reducing.

In the absence of iron films, the addition of Fe (II) is such that N is present₂The O emission is remarkably increased; and in NO₃ ^-Chelation of iron ions on the iron membrane by EDTA in solution significantly reduces N₂And (4) discharging O. These results indicate that Fe (II) oxidation may be coupled with denitrification and promote soil nitrate reduction and N₂Increase in O-emissions (see fig. 5c, 5 d). In the absence of nitrate, Fe (II) gradually decomposes from the iron film, so that the Fe (II) content tends to increase, whereas in the presence of nitrate, iron is coupled by the ferrous oxidation and nitrate reductionThe Fe (II) decomposed on the film is gradually consumed, so that the Fe (II) content tends to gradually decrease or level (see FIG. 5 e).

In summary, compared with CK group, IP group can obviously promote N in hydroponic liquid₂And the concentration is far higher than N₂Emission of O (N under the same experimental conditions)₂In units of μmol L^-1，N₂The concentration unit of O is nmol L^-1By a difference of 10³Fold), the results thus indicate that Fe (II) oxidation may be coupled to denitrification and promote soil nitrate reduction and N₂An increase in emissions. The rice with the induced iron membrane is planted in the soil environment, and the nitrate reduction in the soil environment can be enhanced by the iron membrane treatment, so that the rice rhizosphere soil can generate the denitrification reaction of Fe (II) oxidation coupling. Denitrifying microorganisms and iron redox-related microorganisms are significantly enriched; real-time dynamic monitoring of soil N in iron-coated soil and control-treated soil₂O discharge rate, nitrate, nitrite, ammonium radical concentration, DOC concentration, distribution difference of microbial community structure, solubility N₂The detection data of the O concentration can confirm the conclusion.

Example 2A method for promoting the reduction of nitrate in soil

Transferring rice seedlings to 70mg L^-1And (3) inducing by using a ferrous sulfate solution for 48 hours, transplanting the treated rice seedlings into soil, planting for 4 days, and flooding the soil for 4mm for 9 days.

Example 3A method for promoting nitrate reduction in soil

Transferring rice seedlings to 70mg L^-1And (3) inducing by using a ferrous sulfate solution for 48 hours, transplanting the treated rice seedlings into soil, planting for 5 days, and flooding the soil for 5mm, wherein the flooding time is 10 days.

The results show that the rice planted in the soil can induce the iron film, the microorganisms related to nitrate reduction and iron oxidation reduction are enriched in the artificially regulated environment, and the final product is mainly N₂. These results indicate that the iron membrane conditioning treatment is a nitrate reduction and prevention of ground water in the rhizosphere zone of plantsThe nitrate pollution aspect has a targeted environment-friendly technology.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (1)

1. A method of promoting nitrate reduction in soil comprising the steps of:

applying 10% H to rice seeds₂O₂Soaking the seeds in the solution for 10-15 min for disinfection, thoroughly cleaning the seeds with water, soaking the seeds in the water, and accelerating germination in an incubator;

transplanting the germinated seedlings in the two-leaf period into a rice full nutrient solution for culturing for 18-24d, wherein the nutrient solution is changed every 3d, and the pH value is 5.5-5.8;

planting the four-leaf stage rice seedlings in 70-80 mg L^-1Performing induction culture in a ferrous sulfate solution for 40-48 hours;

after the formation of the iron membrane at the root of the rice is induced, transplanting the rice into soil for culture, and flooding the soil for 5-9 mm for 9-10 d;

wherein the soil contains denitrifying microorganisms and/or iron redox microorganisms coupled with denitrification.

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